The in vivo gene expression signature of oxidative stress.

1Department of Biological Science, University of Tulsa, Tulsa, Oklahoma, USA.

Abstract

How higher organisms respond to elevated oxidative stress in vivo is poorly understood. Therefore, we measured oxidative stress parameters and gene expression alterations (Affymetrix arrays) in the liver caused by elevated reactive oxygen species induced in vivo by diquat or by genetic ablation of the major antioxidant enzymes CuZn-superoxide dismutase (Sod1) and glutathione peroxidase-1 (Gpx1). Diquat (50 mg/kg) treatment resulted in a significant increase in oxidative damage within 3-6 h in wild-type mice without any lethality. In contrast, treatment of Sod1(-/-) or Gpx1(-/-) mice with a similar concentration of diquat resulted in a significant increase in oxidative damage within an hour of treatment and was lethal, i.e., these mice are extremely sensitive to the oxidative stress generated by diquat. The expression response to elevated oxidative stress in vivo does not involve an upregulation of classic antioxidant genes, although long-term oxidative stress in Sod1(-/-) mice leads to a significant upregulation of thiol antioxidants (e.g., Mt1, Srxn1, Gclc, Txnrd1), which appears to be mediated by the redox-sensitive transcription factor Nrf2. The main finding of our study is that the common response to elevated oxidative stress with diquat treatment in wild-type, Gpx1(-/-), and Sod1(-/-) mice and in untreated Sod1(-/-) mice is an upregulation of p53 target genes (p21, Gdf15, Plk3, Atf3, Trp53inp1, Ddit4, Gadd45a, Btg2, Ndrg1). A retrospective comparison with previous studies shows that induction of these p53 target genes is a conserved expression response to oxidative stress, in vivo and in vitro, in different species and different cells/organs.

Liver injury induced by diquat is greater in mice lacking either SOD1 or GPX1

Hepatotoxicity of a single dose of diquat (50 mg/kg body weight given i.p.) was measured by the plasma ALT activity as described in the Experimental Procedures. Graph A: The levels of plasma ALT activity are shown at various times after diquat administration for WT mice. Each point represents the mean ± S.E.M. of data collected from 3 mice. Values with different letter superscripts are significantly different from each other and from the control and 1 h values at the p<0.05 level. Graph B: The effect of genotype and diquat treatment on plasma ALT activity. Plasma ALT activities of WT (open bars), Gpx1−/− (shaded bars), and Sod1−/− (black bars) mice were determined in untreated mice and 1 h after diquat treatment. Each bar represents the mean ± S.E.M. of data from 5 mice. Values with different letter superscripts are significantly different from each other at the p<0.05 level.

Plasma and liver tissue were collected at the indicated time after diquat treatment. The levels of F2-isoprostanes from plasma and liver were determined as described in the Experimental Procedures. The levels of F2-isoprostanes are expressed as ng per ml serum or per g of tissue for isoprostane in plasma and liver, respectively. Graphs A and B: The levels of free F2-isoprostanes were measured in the plasma in WT mice at various times after diquat administration (A) or in the plasma of WT (open bars), Gpx1−/− (shaded bars), and Sod1−/− (black bars) untreated mice and 1 h after diquat treatment (B). Plasma was pooled from 3 mice, and each value represents the mean ± S.E.M. of data from 3 pooled samples (9 mice total). Values with different letter superscripts are significantly different from each other at the p<0.05 level. Graphs C and D: The levels of esterfied F2-isoprostanes were measured in the livers in WT mice at various times after diquat administration (C) or in the livers of WT (open bars), Gpx1−/−(shaded bars), and Sod1−/− (black bars) untreated mice and 1 h after diquat treatment (D). Each value represents the mean ± S.E.M. of data from 4 mice. The “*” indicates a value that is significantly different (p<0.05 level) from untreated mice and mice 12 h after diquat treatment. Values with different letter superscripts are significantly different from each other at the p<0.05 level.

Nuclear DNA was isolated from liver tissue collected at the indicated time after diquat injection and DNA oxidation was measured as described in the Experimental Procedures and expressed as a ratio nmol of 8-oxo-dG to 105nmol of 2-dG. Graph A: The time course of DNA oxidation induced by diquat treatment is shown at various times after diquat administration for WT mice. Each point represents the mean ± S.E.M. of data collected from 4 mice. The “*” indicates values that are significantly different (p<0.05) from untreated mice. Graph B: DNA oxidation was measured in the livers of WT (open bars), Gpx1−/−(shaded bars), and Sod1−/− (black bars) mice before and 1 h after diquat treatment. Each bar represents the mean ± S.E.M. of data from 4 mice. Values with different letter superscripts are significantly different from each other at the p<0.05 level.

The expression pattern elicited by diquat in WT mice is similar to that of the untreated Sod1−/− animals

The fold-change (all fold changes are in comparison to untreated WT control) of genes significantly altered (P<0.005) both by diquat in WT animals (at the 3 or 6 h time points) and in untreated antioxidant knockout mice was determined, and the data are presented graphically. The analysis is restricted to genes altered more than 1.5-fold in both groups. The fold-change in expression in the untreated antioxidant knockout mice (filled diamonds for Sod1−/−, graphs A and C; open diamonds for Gpx1−/−, graphs B and D) is on the y-axis, and that of WT mice treated with diquat is on the x-axis. Data for the 3 h time point are in graphs A and B, and data for the 6 h time point are in graphs C and D. Each symbol represents one specific gene whose x, y coordinates are given by its fold-level expression in antioxidant knockout mice (y) and WT mice treated with diquat (x), in both cases as compared to untreated WT-control mice. A least-square regression line was calculated for each data set, with the slope and R-square values indicated in each graph. There is a statistically significant correlation (P<0.001) for the diquat vs. Sod1−/− comparison but not for the diquat vs. Gpx1−/− comparison (especially note the large number of data points in the lower right quadrant of graph B and D, i.e., genes altered in opposite directions in both groups).

Proteins levels of selected antioxidant genes were measured by Western blots and enzymatic activity (thioredoxin reductase) in Sod1−/− (close bars) and WT mice (open bars). Hemeoxygenase 1 (HO-1), metallothionein (Mt) 1 and 2 (the proteins are too similar to be distinguished by size or antigenicity), thioredoxin-1 (Txn1), peroxiredoxin 1 (Prx-1), glutathione peroxidase 4 (Gpx4), sulfiredoxin (Srxn1), and thioredoxin reductase activity (TxnR) were measured in the cytosolic fraction of the liver. Thioredoxin-2 (Txn2) was measured in the mitochondrial fraction of the liver. The results are the mean of 4-5 animals ± SEM, and the asterisks denote those values significantly different from WT mice at the p<0.05 level.

The nuclear fraction from the livers of Sod1−/− (closed bars) and WT (open bars) mice were analyzed by Western blots as we described in the Experimental Procedures and expressed as a arbitrary units relative to the loading control, histone H1. Graph A: Total Protein levels of p53 and Nrf2 in nuclear extracts. Graph B: Phospho-p53 levels in nuclear and total homogenate extracts. The data are the mean of 3-4 animals ± SEM, and the asterisks denote those values significantly different from WT mice at the p<0.05 level.